beta-arrestin 2 oligomerization controls the Mdm2-dependent inhibition of p53.

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beta-arrestin 2 oligomerization controls the

Mdm2-dependent inhibition of p53.

Cédric Boularan, Mark Scott, Karima Bourougaa, Myriam Bellal, Emmanuel

Esteve, Alain Thuret, Alexandre Benmerah, Marc Tramier, Maité

Coppey-Moisan, Catherine Labbé-Jullié, et al.

To cite this version:

Cédric Boularan, Mark Scott, Karima Bourougaa, Myriam Bellal, Emmanuel Esteve, et al.. beta-arrestin 2 oligomerization controls the Mdm2-dependent inhibition of p53.. Proceedings of the National Academy of Sciences of the United States of America , National Academy of Sciences, 2007, 104 (46), pp.18061-6. �10.1073/pnas.0705550104�. �inserm-00174967�

Supporting Methods.

Fluorescence Lifetime Imaging Microscopy

The two-photon picosecond FLIM system combines multifocal multiphoton excitation (TriMscope, LaVisionBiotec, Bielefeld, Germany) and a fast-gated CCD camera (Picostar, LaVisionBiotec, Bielefeld, Germany) connected to an inverted microscope (IX 71, Olympus, Tokyo, Japan). A mode-locked Ti:Sa laser at 950 nm (Spectra Physics, France) is split up into 2 to 64 beams by utilizing a 50/50 beam splitter and mirrors. The set of beams is passing through a 2000 Hz scanner before illuminating the back aperture of a x60 NA 1.3 infrared water immersion objective (Olympus, Tokyo, Japan). A line of foci is then created at the focal plane, which can be scanned across the sample, making a pseudo wide-field illumination. A filter wheel of spectral filters (535AF45 for GFP) is used to select the fluorescence imaged onto a fast-gated light intensifier connected to a CCD camera (Picostar). The gate of the intensifier is triggered by an electronic signal coming from the laser and a programmable delay box was used to acquire a stack of time-gated images. For each fluorescence lifetime image, a stack of 11 time gated (1 ns gate) fluorescence intensity images at increasing interval after excitation (1 ns per step) was obtained (from 0 to 10 ns), each time gated image being acquired sequentially (from 0 to 10 ns). The acquisition time of the CCD camera was adjusted considering the level of the time integrated fluorescnce signal (from 1 to 4 s). All of the instrumentation was controlled by Imspector software developed by LaVisionBiotec.

To analyze the FLIM data, a mean lifetime image was calculated by using time-correlated stack of images

where ti corresponds to the time delay after the laser pulse of the ith image acquired and Ii to the pixel intensity map in the ith image. Fluorescence lifetime of GFP-fused





Input Flag

IP Myc

IB Flag

Input Myc

IP Myc

IB Myc

IgG heavy chain

Myc -arr2 IP6-C

Myc

-arr2 Flag

Myc -arr2

-arr2 IP6-N Flag

Myc ß-arr2 IP6-N

-arr2 IP6-C Flag

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Supporting Figure 1

Characterization of ß-arr2 IP6-binding mutants

COS7 cells were transfected with the indicated ß-arr2-FLAG and Myc-ß-arr2 constructs or control empty vector (1g of each construct per 100mm dish). After immunoprecipitation with anti-Myc antibodies, immunoblots were performed with anti-Flag and anti-Myc antibodies; 10% of input is shown.

Functional characterization of ß-arr2 IP6-binding mutants in GPCR endocytosis

a) Steady-state localization of ß-arr2 constructs. HeLa cells were transiently

co-transfected with ß-arr2, ß-arr2IP6-N or ß-arr2IP6-C myc-tagged constructs, fixed 48h after transfection (4% PFA) and processed for fluorescence microscopy. b, c) Receptor-dependent accumulation of ß-arr2 constructs in clathrin-coated pits (CCPs) and endosomes. HeLa cells were transiently co-transfected with HA-tagged angiotensin II (AngII) AT1 receptor and either ß-arr2, ßarr2IP6-N or ß-arr2IP6-C YFP-tagged constructs. After 2 (b) or 30 min (c) stimulation with 1M AngII, cells were fixed, permeabilized, and processed for fluorescence microscopy. In the insets, arrows indicate colocalization of arr2 constructs and AP2 in the same CCPs (b) or colocalization of ß-arr2 constructs and transferrin in the same endosomes (c). d) ß-ß-arr2-dependent receptor

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c

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Supporting Figure 3

IP6 binding mutants of -arr2 retain the interaction with AP2 and Filamin A

COS7 cells were transfected with the indicated ß-arr2 constructs alone (a) or in combination with Myc-Filamin A domains 22-24 (b) (1g of each construct per 100mm dish). After immunoprecipitation with anti-Flag antibodies, endogenous AP2 or transfected Filamin A were revealed respectively with anti-AP2 (M300, Santa Cruz) or anti-Myc (AbCam) antibodies; 10% of input is shown.

Myc-Filamin A  -arr2 w t Flag Flag -arr2  IP6-C Flag IP Flag IB Myc Lysates IB Myc IP Flag IB Flag

b

a

AT1AR

0 2 5 15 Time after

Ang II (min) 0 2 5 15 0 2 5 15 Vector -arr2 -arr2 IP6-N

pERK Total

ERK

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a

ERK-scaffolding properties of ß-arr2 IP6-binding mutants

a) Hela cells were transfected with plasmids encoding the HA-tagged angiotensin II

(AngII) AT1 receptor (AT1AR) and ß-arr2 Flag or ßarr2IP6-N Flag or empty vector. After overnight serum starvation, cells were left untreated or stimulated with 1M AngII for 2, 5 or 15 min at 37°C. Cell lysates were resolved by SDS-PAGE and phosphorylation of ERK1/2 was assessed by immunoblotting using an anti-phospho-ERK1/2 antibody. Total ERK1/2 expression was measured by probing the immunoblots with anti-ERK1/2 antibodies. b) Gels were scanned and phospho-and total-ERK1/2 levels determined using the NIH Image J software. The ratio of

 -arr2

-arr2 IP6-N

Supporting Figure 5

b

a

c

d

Vector barr2 Barr2 D Dimbarr2 D Dim

0 25 50 75 % of cells YFP Dapi

-arr2 -arr2IP6-N -arr2IP6-C

Nuclear fluorescence (% of maximum) arr2 arr2 IP6-N arr2 IP6-C Time (min) JNK3 + n=145 n=143 n=179 n=138 -arr2 -arr 2IP6-N -arr 2IP6 -C Vector -arr2-YFP -arr2 IP6-N-YFP -arr2 IP6-C-YFP Jnk3 -arr2 Jnk3

Nucleo-cytoplasmic shuttling of ß-arr2 IP6-binding mutants and JNK3 redistribution a) Nuclear accumulation of ßarr2-YFP, ßarr2 IP6-N-YFP and ß-arr2 IP6-C-YFP 40min

after LMB treatment in Hela cells. b) Kinetics of the nuclear accumulation of ß-arr2-YFP, ß-arr2 IP6-N-YFP and ß-arr2 IP6-C-YFP after LMB addition. c) HeLa cells were transiently co-transfected with ß-arr2, ß-arr2 IP6-N or ß-arr2 IP6-C YFP-tagged constructs and FLAG-JNK3. Cells were fixed, permeabilized, and processed for fluorescence microscopy. The mouse anti-flag M2 monoclonal antibody was revealed using a secondary Cy3-labeled donkey anti-mouse antibody. d) For quantitation, cells were classified respective to their predominantly nuclear, cytosolic or diffuse localization of JNK3. Results are expressed as the percentage of cells in each category. Similar results were obtained in Cos7 cells (data not shown).

Estimation of exogenous -arr in BRET experiments

Western blots comparing the amount of BRET acceptors

expressed in cells displaying half-maximal BRET values in Fig. 2c, compared to the endogenous ß-arrs present in HEK293 and peripheral blood mononuclear cells (PBMC). Equivalent amounts of protein lysates were analyzed using A1CT antibody. To observe transfected BRET acceptors film was exposed for a longer period of time compared to native ß-arrs indicating that the level of expression was at most equivalent to endogenous ß-arr levels.

HEK293 Transf: _ WT IP6 N -arr1 -arr2 IP6 C YFP PBMC WB: -arr (A1CT) Endogenous YFP--arr2

Supporting Table 1

Cell distribution (%) in various phases of the cell cycle

H1299 cells were transfected with the indicated ß-arr2 YFP-tagged constructs alone or in combination with p53 (0,5 g of each construct). The total amount of transfected DNA was kept constant by adding empty vector. 48 h post transfection, H1299 cells nuclei were stained with propidium iodide (50 g/ml) and cell cycle phases were analyzed with a LSR flow cytometer (Becton-Dickinson).